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Microbial Colonization of the Upper Gastrointestinal Tract in Patients with Barrett's Esophagus

  1. Sandra Macfarlane,
  2. Elizabeth Furrie,
  3. George T. Macfarlane, and
  4. John F. Dillon
  1. Dundee University Gut Group, Ninewells Hospital Medical School, Dundee, United Kingdom
  1. Reprints or correspondence: Dr. Sandra Macfarlane, Dundee University Gut Group, Ninewells Hospital Medical School, Dundee DD1 9SY, United Kingdom (s.macfarlane{at}dundee.ac.uk).
 
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Abstract

Background. Barrett's esophagus (BE) is a complication of chronic gastroesophageal reflux disease, in which patients are at greatly increased risk of esophageal dysplasia and adenocarcinoma. Over the past 2 decades, there has been an increase in the incidence of both BE and adenocarcinoma; however, the involvement of microorganisms in BE is uncertain. The aim of this study was to characterize microbial communities in esophageal aspirate specimens and on distal esophageal mucosal samples from patients with BE.

Methods. Biopsy and aspirate specimens were obtained by endoscopic examination from 7 patients with BE and 7 control subjects without BE. Samples were cultured under aerobic, anaerobic, and microaerophilic conditions for yeasts and bacteria, including Helicobacter pylori. Bacterial isolates were identified by 16S ribosomal RNA gene sequencing. Fluorescence microscopic examination was also used to determine the spatial localization of these organisms on mucosal surfaces. Significant colonization was detected in 6 patients with BE and in 4 control subjects.

Results. Overall, 46 bacterial species belonging to 16 genera were detected, with 10 species being common in both groups. Both aspirate and biopsy samples from patients with BE contained complex populations of bacteria. Uniquely, high levels of Campylobacter species (Campylobacter concisus and Campylobacter rectus), which have been linked to enteritis, periodontal infections, and tumor formation in animals, were found in 4 (57%) of 7 patients with BE but in none of the control subjects. Microscopic examination revealed that bacteria on mucosal biofilms often occurred in microcolonies.

Conclusions. The occurrence of nitrate-reducing Campylobacter species in patients with BE may suggest that there is a link in either the initiation, maintenance, or exacerbation of disease processes leading to adenocarcinoma formation.

Barrett's esophagus (BE) is a complication of chronic gastroesophageal reflux disease, in which squamous epithelial cells lining the distal esophagus undergo metaplastic changes, forming a columnar mucosa [1]. Patients with BE have a greatly increased risk of esophageal dysplasia and adenocarcinoma [2, 3]. From 6% to 12% of patients with gastroesophageal reflux disease develop BE [4], and those with BE develop esophageal adenocarcinoma at a rate of ∼1 case per 100 patient-years [4, 5]. Rates of carcinoma of the mid and distal stomach are decreasing; conversely, adenocarcinoma of the esophagus has been steadily increasing in Western countries over the past 3 decades [6, 7], and it is now the seventh most common cause of cancer-related death in the United Kingdom. The number of detected cases of BE is increasing [8], and although all cases of adenocarcinoma are thought to occur in patients with BE, only 1 in 20 patients with adenocarcinoma receive a previous diagnosis of BE. Treatment for adenocarcinoma of the esophagus is generally unsuccessful, with 5-year survival rates as low as 5% [9]. Aggressive therapy for gastroesophageal reflux disease with drugs or antireflux surgical procedures does not prevent BE or progression to cancer [10, 11].

The role of microorganisms in BE is uncertain. The upper gastrointestinal tract, because of intestinal motility and acid secretion, is usually only sparsely colonized by bacteria and yeasts. The number of viable bacterial cells in the stomach is usually <102 cells/mL, with Helicobacter pylori and aciduric gram-positive species, such as lactobacilli and streptococci, usually predominating [12, 13]. Proton pump inhibitors (PPIs) are used to treat patients with gastroesophageal reflux disease; however, this method of treatment can result in microbial overgrowth because of reduced gastric acid and may lead to epithelial damage because of production of nitrosamines and other carcinogens by the bacteria [14, 15]. H. pylori has been shown conclusively to cause duodenal ulcer disease and gastric cancer by indirect mechanisms because of chronic colonization of the superficial gastric mucosa. This bacterium has been investigated in relation to BE, with initial studies indicating that it might have a protective role [16, 17].

In view of the role of H. pylori in upper gut disease, it is possible that other species might be involved in the etiology of BE. To our knowledge, there have been only 2 preliminary studies of bacteria associated with the esophageal mucosa in patients with BE. In the first study [18], it was reported that increased microbial colonization, mainly by gram-positive cocci, occurred during retrospective analysis of stored esophageal tissue from patients with BE in a comparison with healthy control subjects. However, no difference was found on aerobic culture of fresh esophageal biopsy specimens. In the second study, a nonquantitative cloning approach was used to identify the bacteria on a mucosal sample from 1 patient with BE and detected 21 bacterial species, of which ∼50% were categorized as "unidentified" rumen and oral isolates [19].

Because of their close proximity to host tissues, mucosal bacteria are more likely than transient aspirate populations to interact with the esophageal epithelium. Therefore, to determine whether there is a role for mucosal bacteria in BE, it is important to ascertain whether microorganisms form distinct mucosal communities on the epithelial surface or are simply transients resulting from passive transfer from aspirates. Many methods are used to study bacterial colonization, from traditional cultural techniques, using selective plating, to molecular methods, such as fluorescence in situ hybridization [20], denaturing gradient gel electrophoresis [21], real-time PCR [22], and clone library analysis. The advantage of culture techniques is that viable organisms occurring in aspirate specimens or on mucosal surfaces can be quantitated and used for further work.

The aim of this study was to characterize aerobic, microaerophilic, and anaerobic microorganisms in esophageal aspirate specimens and on mucosal surfaces in the distal esophagus of patients with BE and to compare these communities with those in control subjects who have normal esophageal mucosae.

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Patients and Methods

Patients. Esophageal biopsy and aspirate specimens were obtained from 14 individuals (8 females and 6 males aged 36–88 years; mean age, 57 years) attending the Gastroenterology Outpatients Clinic at Ninewells Hospital (Dundee, United Kingdom). Seven subjects had BE, and 7 patients attending the clinic with upper gastrointestinal symptoms requiring endoscopic examination were used as control subjects. The control subjects were shown, by endoscopic and histologic examination, to have no evidence of BE. Patient characteristics are shown in table 1. None of the subjects had received antibiotics within 4 weeks before the investigation. Informed patient consent was obtained from the Tayside Committee on Medical Research Ethics (Dundee, UK).

Enumeration and identification of bacteria in aspirate and mucosal biopsy specimens. Biopsy and esophageal aspirate specimens were transferred to sterile Bijoux bottles containing 4 mL of anaerobic transport medium (Wilkins-Chalgren broth). pH measurements were obtained from a portion of each aspirate specimen. The dimensions of biopsy specimens were determined using a micrometer, after samples had been placed in a anaerobic chamber at 37°C (atmosphere: N2, 80%; CO2, 10%; and H2, 10%). This was used to quantitate bacteria per unit area of mucosal surface. The biopsy specimens were washed gently in the transport medium, placed in 4 mL of fresh transport medium, and dispersed using a sterile glass tissue homogenizer. One milliliter of biopsy homogenate from the biopsy or aspirate specimen was diluted to 10-5 in test tubes containing 9 mL of half-strength anaerobic peptone water. Fifty microliters of the original sample and 100 µL of all dilutions that were diluted to 10-5 were plated in triplicate onto selective and nonselective solid culture media. Aerobes and facultative anaerobes were isolated using Nutrient agar (for total aerobes and facultative anaerobes), Azide Blood agar (for gram-positive cocci), and MacConkey agar (for enterobacteria). Anaerobes were isolated using Wilkins-Chalgren agar with 10% horse blood added (for total anaerobes), Wilkins-Chalgren agar with gram-negative anaerobe supplements (for gram-negative anaerobes), Reinforced Clostridial agar (for clostridia), Azide Blood agar (for anaerobic cocci), and Rogosa agar (for lactobacilli). Bifidobacteria were counted using Beerens agar [23]. The Bacteroides fragilis group were enumerated using Bacteroides Mineral Salts agar [24].

Aerobic plates were incubated at 37°C for 48 h, and anaerobic plates were incubated at 37°C for 5 days. Columbia agar plates supplemented with 5% horse blood, with the addition of Campylobacter species or Helicobacter species selective supplements, were used for selective isolation of Campylobacter species or H. pylori, and chocolate agar plates were used for the isolation of other fastidious microrganisms, such as neisseria and haemophilus. These were incubated at 37°C for 5 days in gas jars containing Gaspaks for microaerophilic or carbon dioxide environments (Don Whitley Scientific). Yeast and Mold agar was used for selective isolation of yeasts. These plates were incubated for 5 days aerobically at 28°C. Bacterial isolates were identified by 16S rRNA gene sequence analysis, and yeasts were identified using the API 20 C AUX biochemical identification system (API, bioMérieux).

DNA extraction from cultures. DNA was isolated using a modified method based on the Qiagen DNA Blood Mini Kit (Qiagen), as described previously [22].

PCR amplification and 16S rRNA gene sequence analysis. Purified DNA was diluted, and 2 µL of the dilution was used as a template in a reaction mix containing 500 nmol/L of eubacterial primers [25], 1.5 mmol/L of MgCl2, 1 mmol/L of Tris-HCL (pH 9.0), 5 mmol/L of KCL, 0.01% of Triton X-100, 1 U of Taq DNA polymerase (Promega), and 250 µmol/L of each dNTP (Bioline) in a total volume of 50 µL. The PCR reactions were denatured at 95°C for 3 min, followed by 35 cycles at 95°C for 1 min, 58°C for 1 min (annealing phase), and 72°C for 45 seconds. The final elongation step was at 72°C for 10 min. Reactions were performed on a Genius Thermal Cycler (Techne). PCR products were resolved by electrophoresis on 1.5% agarose gel containing 0.5 µg/mL of ethidium bromide. Sequencing was performed using Big Dye terminator sequencing on an ABI 3100 Genetic Analyzer (Applied Biosystems). Basic Local Alignment Search Tool searches [26] were performed for each compiled sequence (500 base pairs) against those in the National Center for Biotechnology information database.

Viability staining of bacteria in mucosal tissue. Biopsy specimens were covered with 200 µL of BacLight viability stain (Molecular Probes Europe BV) and visualized, as described previously [20].

Oligonucleotide probes. The 16S rRNA oligonucleotide probes used in the study have been described and validated previously. Probe Str 0493 (5-GTTAGCCGTCCCTTTCTGG-3) was used to identify streptococci [27], and Camp 653 (5-CTGCCTCTCCCTYACTCT-3) was used to identify Campylobacter species [28]. Probes were synthesized by Thermohybaid (Interactiva Division), and were 5′-labeled with FITC or cy3.

Fluorescent in situ hybridization. Hybridization for both probes was performed at 46°C in a humid chamber for 2 h, as previously described [20].

Statistical analysis. Data analysis was performed using GraphPad Prism, version 4.0 (GraphPad Software). Logarithms of microbial numbers were used to achieve normal distribution, and data on the patient groups with and without BE were compared using the independent-sample t test. P < .05 was considered to be statistically significant.

Chemicals. Unless stated otherwise, all bacteriologic culture media and supplements were obtained from Oxoid. Other chemicals were purchased from Sigma.

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Results

Bacteria and yeast measurements. A diverse range of microaerophilic, facultatively anaerobic, and anaerobic gram-positive and gram-negative rods and cocci were detected to be colonizing in esophageal mucosae and aspirate in both control subjects and patients with BE. Aspirate microbiotas were detected in 6 patients with BE and in 4 control subjects. Mucosal colonization occurred in 4 patients with BE and in 3 control subjects. Total counts of bacteria and yeasts in aspirate specimens from patients with BE were higher than those in aspirate specimens from control subjects (figure 1), but the difference was not statistically significant. The number of bacteria and yeasts in mucosal samples from patients with BE were similar to those in mucosal samples from control subjects. PPIs were used to treat all of the patients with BE. Four individuals with healthy esophaguses were not receiving PPIs, and 3 of these persons were colonized by bacteria. The pH of aspirate specimens from patients with BE ranged from 2.0 through 8.0 (mean pH [±SD], 4.5 ± 1.0), and the pH of aspirates specimens from control subjects ranged from 1.6 through 4.0 (mean pH [±SD], 2.3 ± 0.3). Viable bacteria were not detected in any patient samples when the pH of aspirate samples was <2.0, where only yeasts were found. Tables 2–6 show that both aspirate and biopsy materials contained complex bacterial populations, with greater species diversity occurring in the patients with BE. Forty-six bacterial species belonging to 16 genera were found. Eighteen species were isolated from subjects without BE, and 38 species were isolated from patients with BE, with 10 species being common in both groups. H. pylori was not detected in any of the patients.

Figure 1
Figure 1

Total number of bacteria and yeasts isolated from aspirate specimens and esophageal mucosal specimens from 7 control subjects and 7 patients with Barrett's esophagus (BE). The values above the bars represent the number of subjects in whom colonization was detected. Mean numbers and SDs are represented as the tops of the bars and the lines above the bars.

Figure 2
Figure 2

A and B, Confocal sections of mucosal biopsy specimens stained for cell viability, containing mixtures of living (yellow) and dead (red) organisms. Microcolony and aggregate formation can be seen in mucosal samples from control subjects (A) and from patients with Barrett's esophagus (B). C and D, Fluorescence light micrographs of transverse sections of Barrett's esophageal mucosae showing colonization by streptococci (C) and Campylobacter species (D),using 16S ribosomal RNA oligonucleotide probes labeled with FITC and cy3. A, B, and D, Original magnification × 100. C, Original magnification × 60.

Table 1
Table 1

Clinical characteristics of patients in a study of microbial colonization of the upper gastrointestinal tract.

Table 2
Table 2

Gram-positive rods isolated from esophageal aspirate and biopsy material.

Bacteria and yeast populations in control subjects. Lactobacilli, streptococci, and yeasts were the only genera detected in aspirate specimens from patients without BE (tables 2, 3 and 6). Yeasts, when present, reached the highest numbers, and lactobacilli showed the greatest species diversity, with 8 species being identified. In contrast, only 2 Lactobacillus isolates were obtained—together with greater numbers of streptococci—from mucosal tissue from control subjects. No yeasts were detected on the mucosal samples; however, a range of other bacteria, such as actinomyces and prevotella, were found that were not present in the aspirate specimens (tables 2 and 4).

Table 3
Table 3

Gram-positive cocci isolated from esophageal aspirate and biopsy material.

Table 4
Table 4

Gram-negative bacteria isolated from aspirate and esophageal tissue.

Bacteria and yeast populations in patients with BE. A diverse range of bacteria were found in both aspirate and mucosal samples from patients with BE. Lactobacilli were only found in aspirate specimens (table 2). Gram-negative cocci (e.g., veillonella and neisseria) were detected in high numbers in aspirate and mucosal samples from patients with BE (table 4). Prevotellas were only found in the mucosal samples. Yeasts were present in low numbers in 2 aspirate samples and in the mucosal sample from 1 patient. Single isolates of megasphaera, gemella, selenomonas, rothia, and enterobacteria were also found in the aspirate specimens from patients with BE.

Comparison of aspirate and mucosal populations. Higher cell counts and greater species diversity were found in aspirate specimens from patients with BE (13 genera and 28 species) than in aspirate specimens from control subjects (2 genera and 10 species), with 4 species being found in samples from both groups. In particular, more species and increased numbers of streptococci were found in aspirate specimens from patients with BE. Yeasts were present in aspirate specimens from both patient groups, although the number of yeasts was higher in control subjects.

Differences in bacterial genera were found in esophageal mucosal samples from subjects without BE (7 genera and 12 species), compared with mucosal samples from patients with BE (11 genera and 23 species), with 7 species being found in the samples from both groups. Lactobacilli were only detected in mucosal samples from control subjects. Conversely, yeasts, fusobacteria, megasphaera, neisseria, and Camplylobacter species were only isolated from patients with BE. Prevotellas were only detected in mucosal samples in both patient groups, and gram-negative cocci were found in mucosal tissue from 1 control subject and in aspirate and mucosal samples from 4 patients with BE.

Campylobacter species. Uniquely, high levels of Campylobacter species were found to colonize 4 (57%) of 7 patients with BE, but none of the control subjects were colonized by these organisms. Campylobacter species (i.e., Campylobacter rectus and Campylobacter concisus) were found in aspirate samples from 2 patients with BE and in esophageal mucosal samples from 4 patients with BE (Table 5). Campylobacter species were isolated on Wilkins-Chalgren agar with gram-negative anaerobe supplements and incubated anaerobically at 37°C. In patients with BE, C. concisus was the most prevalent pathogen and occurred in the highest numbers. C. rectus was only found in the mucosal samples.

Table 5
Table 5

Campylobacter species isolated from esophageal aspirate and biopsy material from 7 patients with Barrett's esophagus.

Table 6
Table 6

Yeasts isolated from esophageal aspirate and biopsy material.

Microscopic studies. Mucosal bacteria were often found to be growing in microcolonies and cell aggregates, with mucosal samples from patients with BE being more extensively populated. In figure 2A, a bacterial microcolony composed mainly of cocci can be seen on the mucosal sample, whereas in figure 2B, a heterogeneous mixture of different bacteria can be seen spreading over the mucus layer. In figure 2C, large numbers of streptococci can be seen covering the mucosal surface, and colonization by Campylobacter species of a mucosal specimen from a patients with BE is shown in figure 2D.

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Discussion

This article reports, to our knowledge, the first extensive analysis performed on bacteria colonizing mucosal tissue in patients with BE. Our study showed unexpected species diversity in bacterial populations on esophageal epithelia in both patients with BE and control subjects. Distinct differences occurred between mucosal communities and aspirate populations in both patient groups, demonstrating that there is true mucosal colonization by these microorganisms and not just simple aspirate contamination. Bacterial colonization of the upper gut was not entirely dependent on the use of PPIs in the patients with BE, because extensive bacterial growth also occurred in control subjects not receiving PPIs.

Although the columnar-lined epithelium in patients with BE may contain gastric-type epithelium that can be colonized by H. pylori [29], this organism was not detected in our study. To our knowledge, only 2 previous studies have investigated the microbiota in patients with BE. In our investigation, biopsy samples were visualized immediately or stored in a fixative to prevent damage to the bacteria and to preserve tissue architecture. In the study by Osias et al. [18], Gram staining was performed retrospectively on stored tissue samples, and although large numbers of gram-positive cocci were detected on the mucosal samples from patients with BE, this did not take into account potential cell losses during the fixation process. In addition, in our study (figure 2), many mucosal bacteria existed in microcolonies and aggregates and, therefore, could not accurately be quantified by microscopic examination in situ. In the previous investigation [18], when fresh biopsy specimens were cultured, no differences were seen between bacterial counts in patients with BE and counts in control subjects, and only aerobic culture was performed, with no identification of the isolates. In the second investigation by Pei et al. [19], in-depth cloning analysis was performed on 1 patient with BE, and no account was made for aspirate contamination, and the data were not quantitative. In comparison with our study, actinomyces, gram-positive cocci, C. concisus, and C. rectus were not detected in patients with BE.

In our investigation, few consistent differences were observed with regard to esophageal bacterial community structure. A significant exception was the presence of pathogenic nitrate-reducing Campylobacter species in patients with BE. These bacteria were probably not detected previously because they are fastidious, slow-growing microaerophilic organisms that usually require special filtration techniques for isolation [30, 31]. They also need a hydrogen-enriched atmosphere and are inhibited by many of the antibiotics in selective culture media used for isolation of other Campylobacter species, such as Campylobacter jejuni and Campylobacter coli. C. concisus has been associated with periodontal diseases in humans [32, 33] and linked to enteritis in children [30, 31], as well as being associated with diarrheal disease in immunocompromised patients [34]. C. rectus has been implicated as a periodontal pathogen and is found more frequently and at higher levels in diseased sites than in healthy sites in the mouth [35, 36].

There has been speculation that some damage associated with BE is because of nitrate reduction by oral bacteria that form nitrite; under acidic conditions, nitrate reduction can lead to the production of carcinogenic N-nitroso compounds [37,38–39] and nitrous oxide [38, 39], which inhibits DNA repair enzymes and can be mutagenic in high doses [40]. The principal area of nitrite production has been shown to occur at the gastroesophageal junction [41]; this finding lends support to the notion of bacterial involvement in mutagenic events associated with BE. Increased numbers of nitrate-reducing veillonellas were also found in patients with BE, compared with control subjects, and these organisms have been reported to be present in higher levels in oral squamous cell carcinomas [42]. Some strains of C. concisus have been shown to invade Hep2 cells faster than C. jejuni strains [43] and to produce a cytotoxic-like effect on Chinese hamster ovary cells, resulting in intracytoplamic vacuole formation, similar to that caused by H. pylori [44].

Fluorescence microscopic examination demonstrated that many organisms occurred in microcolonies and that aggregates occurred on esophageal tissues (figure 2); live/dead staining showed that the majority of organisms were living. The presence of immunogenic and possibly toxigenic bacterial species in microcolonies on the esophageal epithelium may have implications for BE, because higher localized concentrations of bacterial antigens or waste products, such as nitrite and nitric oxide, would result in a greater degree of tissue damage.

From the results of our study, it is hypothesized that pathogenic and putative toxin-producing Campylobacter species could be involved in either the initiation, maintenance, or exacerbation of esophageal disease processes. A potential link between these organisms and the sequence of events leading from BE to adenocarcinoma formation is intriguing, because not all patients with BE harbor Campylobacter species or develop adenocarcinoma. Studies are now needed to ascertain how Campylobacter species interact with cellular and metabolic processes on the esophageal mucosa and to determine whether they are likely to be involved in the etiology of neoplastic disease.

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Acknowledgments

Financial support. Tenovus (Glasgow, Scotland).

Potential conflicts of interest. All authors: no conflicts.

  • Received January 22, 2007.
  • Accepted March 12, 2007.
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  1. Clin Infect Dis. (2007) 45 (1): 29-38. doi: 10.1086/518578
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